We have identified an imprinted tumor suppressor gene designated ARHI. ARHI encodes a small GTP-binding protein that belongs to the Ras/Rap superfamily, but that has the characteristics of a tumor suppressor gene despite 54-59 % homology to the Ras protooncogenes. ARHI is consistently expressed in normal breast epithelial cells, but is dramatically downregulated in more then 70% of breast cancers. Reexpression of ARHI inhibits growth and induces apoptosis of breast cancer cells. The overall goal of this project is to understand regulation and protein-protein interaction of ARHI in breast cancer. In Specific Aim1, we will identify and characterize the protein complexes that downregulate ARH1 expression in breast cancer cells. We will identify methyI-CpG binding domain protein (MBD) proteins that promote binding of histone deacetylase (HDAC) and histone methyl-transferase (HMT) repressors to the ARHI promoter and negatively regulate gene expression. We will characterize the role of the E2F family proteins on ARHI transcription and their ability to attract HDAC and HMT. In Specific Aim2, we will further characterize the impact of ARHI on Stat3 function. We have found that ARHI interacts directly with Stat3 and inhibits Stat3 activity in breast cancer cells. We will further characterize this novel protein-protein interaction. In Specific Aim3, we will determine the role of ARHI in breast tumorigenesis by knocking down ARHI expression in normal and immortalized human breast epithelial cells. Using the RNAi technology, we will knock down ARHI gene expression to determine whether this will alter the growth of normal human breast epithelial cells or transform immortalized breast epithelial cells. Our Specific Aim 4 is to evaluate ARHI gene therapy in combination with cytotoxic drugs for the treatment of breast cancer. We will attempt to demonstrate additive or synergistic inhibition of breast cancer xenograft growth with a combination of adenovirus-ARHI and cytotoxic drugs through different signaling. These studies will provide a new insight of breast tumorigenesis, and find the potentially useful therapy for breast cancer.